...cells and peroxisomes are where methanol metabolism occurs in methylotrophic organisms. K. phaffii is an ...obligate aerobe. It’s also a methylotropic yeast, meaning it can utilize methanol as its sole source of carbon...media change into methanol and peroxisomal autophagy can be induced by removing methanol from the media....protein animal feed. Due to increasing prices of methanol and advancements in other biomass production techniques...Preferred carbon source Glucose Glucose Glucose, methanol, glycerol Heterothallic mating Efficient...more similarity to the ancient yeast strains and metazoan cells.   Applications for K. phaffii There’s a... its powerful promoter of a gene important for methanol usage. This gene promoter, AOX1, was co-opted ...

...Several methods can be used to create mutational scanning libraries. While each of these methods works ...saturation mutagenesis. Error-prone PCR: This method amplifies the wild-type template with a “sloppy...5) the addition of MnCl2. Disadvantages of this method include favoring of certain types of mutations,...assembly. While this is a comprehensive mutagenesis method, a drawback to using Gibson assembly is that the...PFunkel: Of prior existing comprehensive mutagenesis methods, PFunkel offers the best combination of library...the phage-derived uracil labeled template, this method requires further DNA shuffling to combine pairs...tested in a functional screen of your liking. This method is similar to site-directed mutagenesis (SDM) PCR...

... resolution than using more complex protective methods or detergents like CHAPSO, while requiring a lower...particle orientations, this could be overcome with methods such as chemical crosslinking or tilted-stage imaging... stringent for eVLPs than other viral delivery methods, allowing all the PE components to be delivered...recent publication, the Liu lab showed that this method of delivery could be used to partially correct ...editor consisting of dCas9 fused to a DNA methyltransferase — that can drive lasting transcriptional silencing...Histone tail for Autoinhibition Release of Methyltransferase (CHARM), with several advantages over the ...  Rather than overexpressing an exogenous methyltransferase for epigenetic silencing, which can be toxic...

...acetylation or demethylation. Casilio DNA Methylation Editing (Casilio-ME) is a methylation editing tool...for linking methylcytosine oxidation to DNA repair, which increases activation of methylation-silenced genes...human pluripotent stem cells CRISPR-based DNA demethylation Optogenetic repressors of gene expression ...Using several rapid and cost-effective screening methods, David Liu’s lab identified and validated CBEs ...organism for studying the bacterial cell cycle, asymmetric cell division, and cellular differentiation, ...Find the plasmids Enhanced CRISPR-based DNA demethylation Figure 2: The components of Casilio-ME1...Albert Cheng’s lab that enhances RNA-guided methylcytosine editing by TET1 by co-delivering TET1 and protein...

...multiplexed: you can image over 65 different parameters using this method (see Movie S8 from Radtke et al., 2020...IBEX is a highly multiplexed tissue imaging method that uses iterative staining and chemical bleaching...many conventional microscopes, amplified staining methods, and oligonucleotide-based staining. Of course,..., dye inactivation details, tissue preservation method, and detergents that they used in their experiments...Screenshot showing reagent entries with associated metadata adapted from the Affinity Reagent Working Group...Human Biomolecular Atlas Program (HuBMAP) antibody metadata standards. And the dataset is rich. Experimental... host organism, isotype, clone details, vendor, method, tissue preservation, target tissue, detergent,...

...streptomycin. Mycoplasma can pass through filtration methods because of its ability to change shape and the ...gold standard’ for detecting mycoplasmas. In this method, the supernatant from the cell culture is added...colonies are easily visible on the agar plates. This method is capable of detecting most of the mycoplasma ...mycoplasma species, apart from a few of them. Another method used for mycoplasma detection is using PCR. In this...either species/gene-specific or universal. This method is quick, efficient, reliable and cost-effective...cost-effective (Sung, 2006). The sensor cell-based method uses specialized cells that can detect the presence of ...and this induces a noticeable color change. This method has high sensitivity but low specificity (Degeling...

...nucleotide sequences. To counter this, methyltransferases add methyl groups to bases at specific nucleotide...more stable than the methyltransferase. When a plasmid is lost, the methyltransferase is degraded and/or...the AOX1 promoter whose expression is induced by methanol. Once linearized, the delivery vector can be integrated...Zeocin resistance. Then, counter-selection with methanol medium selects for those cells that have removed... In cells where recombination has not occured, methanol induces the production of the toxic MazF leading...composed of restriction endonucleases and methyltransferases. Restriction endonucleases cleave DNA at ...the cell grows resulting in the appearance of unmethylated sites in the chromosome. The more stable endonuclease...

...how to read a flow plot.  Dot plots Flow cytometry is a method that allows users to analyze single cells...in flow cytometers, and output is generated through measurement of events. An event is something that passes...data in the literature. Data reported from flow cytometry experiments can be a little challenging to understand... read by the machine’s sensors. (Usually this something is a cell, but it could be debris or unbound antibodies...Created with biorender.com.   The goal of flow cytometry is to use increasingly more stringent markers ...in the same order. However, in an actual flow cytometry run, all markers are read in parallel.  Let’s ....  Pro tip! The forward scatter is a proxy for diameter and volume of the cell, thus allowing the exclusion...

...to as “Epi” illumination. Therefore these are sometimes call Epi-fluorescence microscopes. (See left image...enhance performance. In spinning disk microscopy, a metal disk containing a number of holes rotates through...only excites fluorophores within a few hundred nanometers of the glass/water interface. Super resolution...only excites fluorophores within a few hundred nanometers of the coverslip (4). Essentially, an optical...10 fold increase in resolution, down to 10s of nanometers. Thick dynamic samples Ex: 3D cell cultures, ...image thick scattering samples in their entirety, something that is not possible via traditional confocal ... Conchello JA. Fluorescence microscopy. Nature methods 2, 910-919 (2005). PubMed PMID: 16299476. 2. Conchello...

... live cells. Lam et al., Nat Methods 2014 Nov 24. doi: 10.1038/nmeth.3179. Martell et al., Nat Biotechnol...Autophagy 2013 Nov 1;9(11):1852-61. A quicker method for protein inactivation Your protein of interest...advantages of Golden Gate and Multisite Gateway cloning methods developed by the lab of Joachim Wittbrodt might...process provides improvements to classical cloning methods, particularly for complex transgenesis constructs... for certain variants. Klapoetke et al., Nat Methods 2014 Mar 11;(3):338-46. For more information ...system requires less work than using established methods and minimizes some of the technical issues associated...turn RUSH and CRUSH alleles off or on enables this method to rapidly address questions of tissue-specificity...

...Sometimes it feels like DNA and protein get all the attention.There are numerous ways to detect DNA-protein...created the RNA-protein interaction detection (RaPID) method. RaPID borrows the E. coli biotin ligase BirA* ...Challenges with existing RNA-protein interaction methods Not in a native context. While native purification...that the protein of interest interacts with RNA. Methods like cross-linking immunoprecipitation (CLIP) identify...interact with an RNA of interest. Current labeling methods are not ideal for in vivo work. BirA* requires ...RaPID identifies RNA-protein interactions. Both methods rely on the promiscuous E. coli biotin ligase, ...RaPID Like other BirA*-based proximity labeling methods, RaPID cannot distinguish between proteins that...

...some kind of a meta/Frankenstein app? Or perhaps there is a clear winner, is there something that we can ...Examples:  “multiplex design”, “multi-method design”, and “single-method design”. Trends: The most common ...the piñata, their role is to fill the prop with something of value. They are reviewers of content. On the...and its contents spill to the ground. There is something akin to a frenzy as the crowd tries to weigh and...what the reviewers so painstakingly assessed to something a bit more representative of the communities’ ... combination of both empirical and approximate methods. The details of their scoring function can be found... al., 2013, other tools have incorporated new parameters such as the Doench-Root score (by Doench et al...

...but what about the other methods to quantify DNA? Here we cover five methods to do this. 5. Plasmids ...depositors who quickly shared their reagents: detection methods, synthetic nanobodies, SARS-CoV-2 protein expression...to quantify AAV titers. Get the details on the methods in this blog post. 8. Chromoproteins: Colorful...They've been adapted for many molecular biology methods such as allelic exchange, FRET, and synthetic biology...Quantifying DNA? Here are Five DNA Quantification Methods to Consider by Jennifer Tsang The nanodrop vs...without a kit. 3. SARS-CoV-2/COVID-19 Detection Methods Based on CRISPR/Cas by Shravanti Suresh Guest ... CRISPR at Iowa State University detailed five methods for using CRISPR to detect SARS-CoV-2, including...

...different PCR methods have been developed for different scientific applications. These PCR methods all use ...PCR   Type of PCR Goal of the PCR method Uses of PCR method End point PCR or just PCR Amplify...polymerase chain reaction (PCR). PCR is an in vitro method in which a small amount of DNA can be copied many...extremely useful but is not as quantitative as other methods of PCR. Theoretically scientists should be able...intercalates with any dsDNA and the use of a fluorometer feature built into the thermocycler to measure...droplet PCR, ddPCR  Digital droplet PCR or ddPCR is a method that provides ultrasensitive and absolute nucleotide...Multiplex PCR Multiplex PCR, as the name implies, is a method in which multiple targets can be amplified in a...

... of this method, isolation of a specific genomic region was an intensive process. One method required ..., the Hodaka Fujii Laboratory first described a method to purify a specific genomic region using the CRISPR...this site (Fujita and Fujii, 2013). They call this method engineered DNA-binding molecule-mediated chromatin...the identification of DNA binding proteins, this method can characterize nucleic acid-nucleic acid interactions...2018). In addition to the "in cell" form of this method, in which an engineered DNA-binding molecule such...purification schemes add additional flavors to this methodology. Using this technique, researchers have begun... combined with next-generation sequencing, this method allows for the recovery of a vast amount of data...

...cloning method and its advantages. An introduction to Gateway technology The Gateway cloning method, developed...entry clone - human KRAS flanked by attL sites.  Method A: recombination of an attB-PCR product or plasmid...to design Gateway plasmids.    Figure 3: Method A to create an entry clone: recombination of an...product with an attP-containing donor vector.   Method B: TOPO-cloning of the desired insert into an attL-entry-TOPO...attL-containing entry vector.   Figure 4: Method B to create an entry clone: TOPO cloning the insert...insert into an attL-containing entry vector.   Method C: Restriction cloning of a restriction enzyme ...attL-containing entry vector.   Figure 5: Method C to create an entry clone: Restriction cloning...

...There are multiple methods for fixing samples, each with its own pros and cons. The method you use will depend...To narrow down which specific methods to use, start by reading the methods sections of papers that have...validation of antibodies. Nat Methods 13:823–827. https://doi.org/10.1038/nmeth.3995 Additional Resources ... to assays using non-fluorescent visualization methods. Stay tuned for a future blog post to learn more...will be able to image them in the end. Again, the methods you use will depend on target and sample type. ... fluorescence and to capture your results. The methods used depend on the sample type, microscopy tools...to Performing Immunofluorescence Staining. In: Methods in Molecular Biology. Springer New York, pp 299...

...comes to labeling cells for flow cytometric analysis, the most common method is a cell surface label, where...single-fluorophore-conjugated antibodies. These methods can include labeling markers with multiple proteins... cannot be used, you may want to look at other methods. In the above cases, a good alternative is using... used in western blotting or ELISAs.  A popular method for indirect labeling employs a primary epitope-targeting...the cells that are not labeled. An easy and quick method of negatively selecting your cells of interest .... The main applications of dye stains in flow cytometry are cell proliferation and cell viability. Unlike...(histogram) flow plot (Figure 4b). Through this method, differences in proliferation between cell populations...

... and mass spectrometry approaches. Recently however, scientists have developed newer methods termed proximity-based...antibody based affinity purification/mass spectrometry methods.  BioID: a promiscuous biotin ligase that...purified using streptavidin and separated via flow cytometry.  Find plasmids for EXCELL here! Since BioID was...your favorite protein or RNA motif, give these methods a try.  References:  Branon, Tess C., et al. ...Khavari. "Research Techniques Made Simple: Emerging Methods to Elucidate Protein Interactions through Spatial...electron microscopy and proximity labeling." Nature methods 12.1 (2015): 51. PubMed PMID: 25419960. PubMed ...interaction detection in living cells." Nature methods 15.3 (2018): 207. PubMed PMID: 29400715. PubMed...

...normal neuronal context. In other words, I needed a method for visualizing protein turnover in situ in neurons... come with health risks. Importantly, with this method it is not possible to follow turnover of individual...overexpression may interfere with the system. Another method for studying protein synthesis in situ is the knock-in... FUNCAT Professor Schuman’s technique applies metabolic labeling using a noncanonical amino acid, azidohomoalanine...incorporated into newly synthesized proteins instead of methionine. The labeled proteins can be detected using click-chemistry...originally published by the Söderberg lab in Sweden as a method for the visualization of protein-protein interactions... later visualized by FUNCAT-PLA. Before these methods were combined, it was not possible to visualize...